While many genome sequencing projects are now complete and it is possible to create complete lists of all protease genes in an entire organism, our understanding of the functional roles of these enzymes, for the most part, remains largely unclear. The development of new technologies that will allow the imaging of protease activity is a critical step to begin to map out complex proteolytic cascades. This proposal outlines plans to develop small molecule activity based probes (ABPs) for functional microscopic imaging of two major classes of cystiene proteases, the cathepsins and caspases. Specifically we plan to 1) develop both general and selective cysteine protease probes that will modify protease targets in complex proteomes 2) develop fluorescently labeled probes that become activated upon modification of a target enzyme and 3) validate the probes using mouse models for ischemia-induced cell death and VEGF-mediated angiogenesis. We plan to use both invasive microscopic methods for high resolution imaging as well as non-invasive methods for direct imaging of protease activities in live animals. Thus, chemical probes have broad applications for basic mechanistic studies as well as for diagnostic imaging in a number of human diseases in which cysteine proteases play a functional role. [unreadable] [unreadable]